Interferon for treating or preventing a coronaviral infection

ABSTRACT

The present invention provides a composition and method for use in the prevention or treatment of a coronaviral infection and in particular, the human coronavirus infection termed severe acute respiratory syndrome (SARS) coronavirus (SARS-HCoV). A method of treating a coronaviral infection is provided through the administration of interferon, further the use of interferons in the treatment of a coronaviral infection is also provided. Preferred forms of interferon for use in the invention are multi-subtype interferon products such as multi-subtype, human alpha-interferon derived from white blood cells commercially available as Multiferon.

FIELD OF THE INVENTION

The present invention provides a composition for use in the treatment orprevention of a coronavirus infection, more specifically a humancoronaviral infection, most specifically severe acute respiratorysyndrome (SARS) coronavirus.

BACKGROUND OF THE INVENTION

Viral Infection

Viral infection is initiated by the binding of a viral particle to areceptor on the surface of a host cell membrane. The virus passes intothe cell by endocytosis. Enzymes encoded by the viral genome aretranscribed by the host cell and cause the viral coat to fuse with theendosome membrane causing the viral genome to be released into thecytosol. The virus uses the host cell to effect protein production inorder to make numerous copies of the genome. Viral coats are formed fromcoat proteins encoded by the viral genome and synthesised by host cellribosomes. The viral genomes are then packaged into the newly producedviral coats and expelled from the host cell via the intracellularprotein trafficking pathway or through cell lysis. The newly synthesisedviral particles are then available for infection of other host cells.

Coronaviruses

Members of the order Nidovirales, the coronaviruses are enveloped,single stranded RNA viruses. Coronoviral infection causes severe diseaseof the respiratory and enteric systems. Coronaviruses have beenassociated with gastroenteritis, hepatitis, peritonitis and bronchitis.However, infection in humans generally results in milder symptoms. TheSARS human coronavirus (SARS-HCoV) appears to be the first coronaviruswhich regularly causes severe disease in humans. SARS-HCoV causes severepneumonia-like symptoms in those infected, with mortality occurring inthe most severe cases.

Treatment of SARS

Various anti-viral treatments have been administered to humans infectedwith SARS-HCoV, including general anti-virals, treatments which inhibitviral cell entry or replication, and immunostimulants.

Ribavirin is a broad spectrum anti-viral agent based on a purinenucleoside analogue and is the standard treatment regimen for hepatitisC. Ribavirin is known to be active against various RNA viruses byinducing lethal mutagenesis of the viral RNA genome (Crotty et al.,2000; Tam et al., 2001) and is known to show anti-viral activity againstanimal coronaviruses (Weiss & Oostrom-Ram, 1989; Sidwell et al., 1987).However, in vitro tests of the efficacy of the drug against SARS-HCoVhave produced a series of negative results and adverse reactions havealso been reported.

A limited number of other drugs have undergone testing. The influenzadrug, Oseltamivir, a neuramidase inhibitor, has undergone analysis forits efficacy against SARS-HCoV infection, but has not shown anytherapeutic benefit (Lee et al., 2003 and Poutanen et al., 2003). Inlaboratory tests, Cystatin C, a protease inhibitor found in human blood,was found to block replication of the ‘common cold’ coronaviruses, butthis has not been tested against SARS-HCoV. It is unlikely that CystatinC will be a candidate for the treatment of SARS-HCoV infected patients,since it has not undergone the safety and efficacy tests required forall human therapeutics.

Interferons

The interferons (IFNs) may be classified into two distinct types—Type IIFNs and the Type II IFNs. The type I IFNs consist of IFN alpha and IFNbeta, whereas the Type II group consists of IFN gamma. Type I IFNs areproduced in direct response to a viral infection.

IFN alpha is represented by a large family of structurally related genesexpressing at least thirteen subtypes, whereas IFN beta is encoded by asingle gene (Diaz et al., 1996). Both types of IFN are able to stimulatean anti-viral state in target cells, whereby the replication of a virusis inhibited through the synthesis of enzymes which interfere with thecellular and viral processes.

Type I IFNs also act to inhibit or slow the growth of target cells andmay render them more susceptible to apoptosis. This has the effect oflimiting the extent of viral spread. Type I IFNs are immunomodulators,or ‘biological response modifiers’ which act to stimulate the immuneresponse. Even though IFN alpha and IFN beta show many broadsimilarities in their actions, there are significant differences in themanner by which they exert their effects and it is these extendedfunctions that account for the different ranges of antiviral activitiesof the two types. A review of the different mechanisms by whichinterferons exert their anti-viral effects is provided by Goodbourn etal., 2000.

Recombinant interferons, which consist of only the IFN alpha 2 subtype,currently dominate the market for anti-viral and oncology indications.The two main recombinant alpha IFN products, Intron A™ from ScheringPlough (IFN-alpha 2b) and Roferon™ (IFN-alpha 2a) from Roche. Incontrast to these single-subtype products, there are several alpha IFNpreparations that consist of a mixture of different subtypes. Thesemulti-subtype IFN alpha products are produced either by human leukocytesin response to a stimulation from a virus (such as Multiferon™ fromViragen, Inc or its subsidiaries, or Alferon-N™ from InterferonSciences/Hemispherix), or in human lymphoblastoid cells, cultured from apatient with Burkitt's lymphoma (such as Sumiferon™ from Sumitomo).

There are many differences between the recombinant forms of IFN alphaand the multi-subtype forms. The most obvious difference is the numberof IFN alpha subtypes each possesses. As mentioned previously, therecombinant forms comprise only the alpha 2 subtype—the alpha 2b formfor Intron A™ (Schering Plough) and the alpha 2a form for Roferon™(Roche). These two allelic variants differ by only one amino acidresidue. The multi-subtype forms of IFN alpha, as the name suggests,comprise many subtypes of IFN alpha. Another difference between themulti-subtype and the recombinant forms is that the IFN alpha 2 producedby human cells in the manufacturing process of the multi-subtype formsis glycosylated, whereas the recombinant forms are unglycosylated, inthat they are produced through bacterial fermentation. Glycosylationplays a major role in many functions of the protein product, such ashalf-life, the bioactivity and its immunogenicity. Therefore, theglycosylation of a product is an important consideration when developinga therapeutic or prophylactic treatment, as it may affect the durationin the body after administration, the activity of a therapeuticallyappropriate dose and the tolerability to the product itself.

During the last decade, considerable progress has been achieved in theidentification of the components, as well as the molecular eventsinvolved in the immunotherapeutic effects of interferons. Over thirtydifferent proteins have been identified that have been shown to beinduced by interferon (Strannegard, 2002, unpublished review).

There are currently no completely effective therapeutic or prophylactictreatments for humans infected with coronavirus and in particularSARS-HCoV. There thus exists a need for an effective treatment forcoronaviral infection in humans, and in particular for severe acuterespiratory syndrome (SARS) coronavirus.

SUMMARY OF THE INVENTION

The present inventors have now shown that interferons and in particularmultiple subtype natural human alpha interferon products aresurprisingly effective at treating human coronavirus infection, and inparticular severe acute respiratory syndrome (SARS) coronavirus.

According to a first aspect of the present invention there is provided amethod of treating coronaviral infection, the method including the stepof administering a therapeutically useful amount of an interferon to asubject in need of treatment.

In one preferred embodiment, the method of treatment can be used toprevent coronaviral infection, the method including the step ofadministering a therapeutically useful amount of an interferon to asubject sufficient to cause protection against infection.

Interferon in each or any of the aspects of the invention is preferablyisolated interferon. An isolated interferon is an interferon which issynthetic (e.g. recombinant), or which is altered, removed or purifiedfrom the natural state through human intervention. For example, aninterferon naturally present in a living animal is not isolated, whereasa synthetic interferon, or an interferon which is partially orcompletely separated from the coexisting materials of its natural state,is isolated. An isolated interferon can exist in substantially purifiedform, or can exist in a non-native environment such as, for example, acell into which the interferon has been introduced. Interferons purifiedfrom human cells, for example the multi-subtype, human alpha-interferonderived from white blood cells commercially available as Multiferon™from Viragen, Inc. or any of its subsidiaries, are also considered to beisolated molecules for purposes of the present invention.

The interferon may be any suitable interferon, for example interferonalpha or interferon beta. It may be single or multi-subtype, but ispreferably multi-subtype.

The interferon may be naturally derived, for example from human cells orrecombinant, but preferably the interferon is naturally derived.Preferably the naturally derived interferon is obtained from leukocytesfollowing viral stimulation or produced in human lymphoblastoid cellscultured from a patient with Burkitt's lymphoma.

Preferred interferons for use in the invention include multi-subtypeinterferon alpha (IFNα), interferon αn1, interferon αn3 or interferonβ1b. A particularly preferred interferon for use in the invention is themulti-subtype IFNα product commercially available from Viragen, Inc. orany of its subsidiaries under the trade name Multiferon™.

As used herein the term Multiferon™ refers to a highly purified,multi-subtype, human alpha interferon derived from human white bloodcells commercially available from Viragen, Inc or any of itssubsidiaries.

According to a second aspect of the present invention there is providedan interferon for use in the treatment or prevention of a coronaviralinfection.

Preferably the interferon is an isolated interferon.

The interferon may be any suitable interferon, for example interferonalpha or interferon beta. It may be single or multi-subtype, but ispreferably multi-subtype.

The interferon may be naturally derived, for example from human cells orrecombinant, but preferably the interferon is naturally derived.Preferably the naturally derived interferon is obtained from leukocytesfollowing viral stimulation or produced in human lymphoblastoid cellscultured from a patient with Burkitt's lymphoma.

Preferred interferons for use in the invention include multi-subtypeinterferon alpha (IFNα), interferon αn1, interferon αn3 or interferonβ1b. A particularly preferred interferon for use in the invention is themulti-subtype IFNα product commercially available from Viragen, Inc. orany of its subsidiaries under the trade name Multiferon™.

As used herein the term Multiferon™ refers to a highly purified,multi-subtype, human alpha interferon derived from human white bloodcells commercially available from Viragen, Inc or any of itssubsidiaries.

According to a third aspect of the present invention there is providedthe use of an interferon in the preparation of a medicament for thetreatment or prevention of a coronaviral infection.

Preferably the interferon is an isolated interferon.

The interferon may be any suitable interferon, for example interferonalpha or interferon beta. It may be single or multi-subtype, but ispreferably multi-subtype.

The interferon may be naturally derived, for example from human cells orrecombinant, but preferably the interferon is naturally derived.Preferably the naturally derived interferon is obtained from leukocytesfollowing viral stimulation or produced in human lymphoblastoid cellscultured from a patient with Burkitt's lymphoma.

Preferred interferons for use in the invention include multi-subtypeinterferon alpha (IFNα), interferon αn1, interferon αn3 or interferonβ1b. A particularly preferred interferon for use in the invention is themulti-subtype IFNα product commercially available from Viragen, Inc. orany of its subsidiaries under the trade name Multiferon As used hereinthe term Multiferon™ refers to a highly purified, multi-subtype, humanalpha interferon derived from human white blood cells commerciallyavailable from Viragen, Inc or any of its subsidiaries.

Preferably the coronaviral infection is a human coronaviral infection.Most preferably the coronaviral infection is severe acute respiratorysystem (SARS) coronavirus (SARS-HCoV).

According to a fourth aspect of the present invention there is provideda method of treating or preventing human infection with a coronavirus,and in particular severe acute respiratory system (SARS) coronavirus(SARS-HCoV), the method including the step of administering atherapeutically useful amount of an interferon to a subject in need oftreatment along with a therapeutically useful amount of a suitableanti-viral compound.

In one preferred embodiment, the method of treatment includes theprevention of human infection with a coronavirus, wherein the methodincludes the step of administering a therapeutically useful amount of aninterferon, or administering an amount of an interferon along with anamount of a suitable anti-viral compound sufficient to cause protectionagainst the infection.

Preferably the interferon is an isolated interferon.

Preferably the anti-viral compound is ribavirin.

Preferably the interferon is any suitable interferon, for exampleinterferon alpha or interferon beta. It may be single or multi-subtype,but is preferably multi-subtype.

The interferon may be naturally derived, for example from human cells orof recombinant form, but preferably the interferon is naturally derived.Preferably the naturally derived interferon is obtained from leukocytesfollowing viral stimulation or produced in human lymphoblastoid cellscultured from a patient with Burkitt's lymphoma.

Preferred interferons for use in the invention include multi-subtypeinterferon alpha (IFNα), interferon αn1, interferon αn3 or interferonβ1b. A particularly preferred interferon for use in the invention is themulti-subtype IFNα product commercially available from Viragen, Inc. orany of its subsidiaries under the trade name Multiferon™.

As used herein the term Multiferon™ refers to a highly purified,multi-subtype, human alpha interferon derived from human white bloodcells commercially available from Viragen, Inc or any of itssubsidiaries.

According to a fifth aspect of the present invention there is providedthe use of interferon and an anti-viral compound in the preparation of acombined medicament for the treatment or prevention of infection with acoronavirus, and in particular severe acute respiratory system (SARS)coronavirus (SARS-HCoV).

Preferably the interferon is an isolated interferon.

Preferably the anti-viral compound is ribavirin.

Preferably the interferon is any suitable interferon, for exampleinterferon alpha or interferon beta. It may be single or multi-subtype,but is preferably multi-subtype.

The interferon may be naturally derived, for example from humans cell,or of recombinant form, but preferably the interferon is naturallyderived. Preferably the naturally derived interferon is obtained fromleukocytes following viral stimulation or produced in humanlymphoblastoid cells cultured from a patient with Burkitt's lymphoma.

Preferred interferons for use in the invention include multi-subtypeinterferon alpha (IFNα), interferon αn1, interferon αn3 or interferonβ1b. A particularly preferred interferon for use in the invention is themulti-subtype IFNα product commercially available from Viragen, Inc. orany of its subsidiaries under the trade name Multiferon™.

As used herein the term Multiferon™ refers to a highly purified,multi-subtype, human alpha interferon derived from human white bloodcells commercially available from Viragen, Inc or any of itssubsidiaries.

The term ‘treatment’ as used herein refers to any regime that canbenefit a human or non-human animal. The treatment may be in respect ofan existing condition or may be prophylactic (preventative treatment).Treatment may include curative, alleviation or prophylactic effects.

Administration

Interferons of and for use in the present invention may be administeredalone, or in combination with another agent, but will preferably beadministered as a pharmaceutical composition, which will generallycomprise a suitable pharmaceutical excipient, diluent or carrierselected dependent on the intended route of administration.

Interferons of and for use in the present invention may be administeredto a patient in need of treatment via any suitable route. The precisedose will depend upon a number of factors, including the precise natureof the interferon.

Some suitable routes of administration include (but are not limited to)oral, rectal, nasal, topical (including buccal and sublingual), vaginalor parenteral (including subcutaneous, intramuscular, intravenous,intradermal, intrathecal and epidural) administration, or administrationvia oral or nasal inhalation.

In preferred embodiments, the composition is deliverable as aninjectable composition, is administered orally, is administered to thelungs as an aerosol via oral or nasal inhalation.

For administration via the oral or nasal inhalation routes, preferablythe active ingredient will be in a suitable pharmaceutical formulationand may be delivered using a mechanical form including, but notrestricted to an inhaler or nebuliser device.

Further, where the oral or nasal inhalation routes are used,administration by a SPAG (small particulate aerosol generator) may beused.

For intravenous injection, the active ingredient will be in the form ofa parenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally comprise a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

The composition may also be administered via microspheres, liposomes,other microparticulate delivery systems or sustained releaseformulations placed in certain tissues including blood. Suitableexamples of sustained release carriers include semipermeable polymermatrices in the form of shared articles, e.g. suppositories ormicrocapsules. Implantable or microcapsular sustained release matricesinclude polylactides (U.S. Pat. No. 3,773,919 and European PatentApplication Publication No 0,058,481) copolymers of L-glutamic acid andgamma ethyl-L-glutamate (Sidman et al., Biopolymers 22(1): 547-556,1985), poly(2-hydroxyethyl-methacrylate) or ethylene vinyl acetate(Langer et al., J. Biomed. Mater. Res. 15: 167-277, 1981, and Langer,Chem. Tech. 12:98-105, 1982, the entire disclosures of which are hereinincorporated by reference).

Examples of the techniques and protocols mentioned above and othertechniques and protocols which may be used in accordance with theinvention can be found in Remington's Pharmaceutical Sciences, 16thedition, Oslo, A. (ed), 1980, the entire disclosure of which is hereinincorporated by reference

Pharmaceutical Compositions

As described above, the present invention extends to a pharmaceuticalcomposition for the treatment or prevention of a coronaviral infection,wherein the composition comprises at least one interferon.Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention may comprise, in additionto active ingredient (i.e. one or more interferons), a pharmaceuticallyacceptable excipient, carrier, buffer stabiliser or other materials wellknown to those skilled in the art. Such materials should be non-toxicand should not interfere with the efficacy of the active ingredient. Theprecise nature of the carrier or other material will depend on the routeof administration, which may be, for example, oral, intravenous, orintranasal.

The formulation may be a liquid, for example, a physiologic saltsolution containing non-phosphate buffer at pH 6.8 to 7.6, or alyophilised powder.

Dose

The composition/interferon is preferably administered to an individualin a “therapeutically effective amount”, this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc, is ultimately within the responsibility and at thediscretion of general practitioners and other medical doctors, andtypically takes account of the disorder to be treated, the condition ofthe individual patient, the site of delivery, the method ofadministration and other factors known to practitioners.

The optimal dose can be determined by physicians based on a number ofparameters including, for example, age, sex, weight, severity of thecondition being treated, the active ingredient being administered andthe route of administration.

For example, in one embodiment, a suitable dose of interferon may be 1to 10 million IU, for example 3 to 5 million IU three times weekly to0.5 to 10 million, for example 2 to 8 million, or 4 to 6 million IUdaily, although other doses may be used.

According to a further aspect of the present invention there is providedan assay method for determining the efficacy of a composition in thetreatment or prevention of a coronaviral infection, wherein thecomposition comprises an interferon, preferably a multi sub-typeinterferon.

In a further aspect of the present invention, there is provided an assaymethod for determining the efficacy of a candidate agent in thetreatment of a coronaviral infection, wherein the assay method includesthe steps of;

-   -   incubating virus infected cells in the presence of the candidate        agent, and    -   determining the degree of inhibition of the cytopathic effect of        the virus on the cells.

Preferably the method includes the further step of comparing the degreeof viral inhibition obtained using the candidate agent with the degreeof viral inhibition obtainable with incubation with an interferon orinterferon based product.

Preferably the interferon is a multi-subtype interferon, most preferablyMultiferon™.

In a still further aspect, there is provided an assay method fordetermining the efficacy of a candidate agent in the prevention of acoronaviral infection, wherein the assay method includes the steps of:

incubating cells in the presence of the candidate agent,

adding the coronavirus to the cells, and

determining the degree of protection against the coronaviral infectionafforded by the candidate agent

Preferred assays for use in the assay methods of the invention includecytopathic endpoint assays and plaque reduction assays.

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis unless the context demands otherwise.

Unless otherwise defined, all technical and scientific terms used hereinhave the meaning commonly understood by a person who is skilled in theart in the field of the present invention

Throughout the specification, unless the context demands otherwise, theterms ‘comprise’ or ‘include’, or variations such as ‘comprises’ or‘comprising’, ‘includes’ or ‘including’ will be understood to imply theinclusion of a stated integer or group of integers, but not theexclusion of any other integer or group of integers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described with reference to thefollowing examples which are provided for the purpose of illustrationand are not intended to be construed as being limiting on the presentinvention, and further, with reference to the figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a dose response curve produced from an in vitro plaquereduction assay, showing that with increasing concentrations of theMultiferon™, the effect of the SARS-HCoV virus is attenuated;

FIG. 2 shows the effect of Multiferon™ and Intron A™ on thecytopathogenicity of Semliki Forest Virus (SFV) on African Green MonkeyKidney Vero E6 cells;

FIG. 3 shows the effect of Multiferon™ on the cytopathogenicity of humanEncephalomyocarditis virus (EMCV) on human A459 cells, wherein theMultiferon™ concentration required to obtain 50% cytopathic effect (CPE)for human A459 cells challenged with EMC virus is shown for differentconcentrations of EMC virus, presented as a 1/dilution; and

FIG. 4 shows the effect of increasing concentrations of Multiferon™ onhuman A459 cell survival. Cell survival was measured photometrically atAbs_(595 nm) using a fixed dilution of EMC virus (dilution 1/400), atincreasing concentrations of Multiferon. AU denoted Absorbance Units.

EXAMPLES Example 1 Anti-Viral Effect of Interferon Against SARS-HCoVInfection in Vero E6 Cells

The effectiveness of the interferons to inhibit the cytopathic effectfollowing SARS-HCoV infection was tested in a cytopathic endpoint assayand a plaque reduction assay. All endpoint assays were carried out usingthe multi-subtype interferons Multiferon™ and interferon αn3, as well assingle subtype recombinant interferon alpha (subtypes interferon α2a,interferon α2b, and interferon αn1) and the interferon beta (IFNβ)subtypes interferon β1a and interferon β1b as well as the anti-viralRibavirin for comparison.

Preparation of Anti-Viral Treatments

A broad range of concentrations (obtained by ten-fold dilutions)encompassing the inhibitory dosages stated by the manufacturer for otherviral-host combinations was tested. Compounds already present in aqueousinjections were made up to volume using Hank's buffered saline solution.For tablet and capsule formulations with soluble active ingredients, theouter coat was removed wherever applicable and the preparation ground ina mortar and pestle. The contents were dissolved in water, vortexed andcentrifuged thereafter at 3000 G. The required volume was pipetted fromthe supernatant and diluted accordingly. Where active ingredients wereinsoluble in water, the contents were dissolved in dimethylsulphoxide(DMSO) and care was taken to ensure that the final concentration of DMSOin the dilutions would not exceed 1%. For plaque assays, 5-fold drugdilutions were prepared using growth media as specified below.

SARS-HCoV Production and Infection

African Green Monkey (Vero E6) cells (American Type Culture Collection,Manassas, Va., USA) were propagated in 75 cm² cell culture flaskscontaining growth medium consisting of medium 199 (Sigma, St Louis, USA)supplemented with 10% foetal calf serum (FCS; Biological Industries,Israel). SARS-HCoV 2003VA2774 (an isolate from a SARS patient inSingapore) was propagated in Vero E6 cells. Briefly, 2 ml of stock viruswas added to a confluent monolayer of Vero E6 cells and incubated at 37°C. in 5% CO₂ for one hour. 13 ml of medium 199 supplemented with 5% FCSwas then added. The cultures were incubated at 37° C. in 5% CO₂ and theinhibition of cytopathic effect gauged by observing each well through aninverted microscope. Where 75% or greater inhibition was observed after48 hours, the supernatant was harvested. The supernatant was clarifiedat 2500 rpm and then aliquoted into cryovials and stored at −80° C.until use.

Virus Handling and Titration

Virus titre in the frozen culture supernatant was determined using aplaque assay carried out in duplicate. Briefly, 100 microlitres of virusin 10-fold serial dilution was added to a monolayer of Vero E6 cells ina 24 well-plate. After incubation for an hour at 37° C. in 5% CO₂, theviral inoculum was aspirated and 1 ml of carboxymethylcellulose overlaywith medium 199 supplemented with 5% FCS was added to each well. Afterfour days of incubation, the cells were fixed with 10% formalin andstained with 2% crystal violet. The plaques were counted visually andthe virus titre in plaque forming units per ml (pfu/ml) calculated.

Cytopathic Endpoint Assay

The protocol used was adapted from Al-Jabri et al. 1996. The effect ofeach anti-viral treatment was tested in quadruplicate. Briefly, 100microlitres of serial 10-fold dilutions of each treatment were incubatedwith 100 microlitres of Vero E6 cells giving a final cell count of20,000 cells per well in a 96-well plate. Incubation was at 37° C. in 5%CO₂ overnight for the interferon preparations and for one hour forRibavirin. 10 microlitres of virus at a concentration of 10,000 pfu/wellwere then added to each test well. This equates to a multiplicity ofinfection (MOI) (virus particles per cell) of 0.5. The plates wereincubated at 37° C. in 5% CO₂ for three days and the plates wereobserved daily for cytopathic effects. The end point was the dilutedconcentration that inhibited the cytopathic effect in all four set-ups(CIA₁₀₀).

To determine cytotoxicity, 100 microlitres of serial 10-fold dilutionsof each treatment were incubated with 100 microlitres of Vero E6 cellsgiving a final cell count of 20,000 cells per well in a 96-well plate,without viral challenge. The plates were then incubated at 37° C. in 5%CO₂ for three days and toxicity effects were observed for using aninverted microscope.

Interferons which showed complete inhibition were tested further at thelower viral titres of 10³ and 10² pfu/well.

Plaque Reduction Assay

Multiferon™, interferon αn3 and interferon β1b were further tested usinga plaque reduction assay. Trypsinised Vero E6 cells were re-suspended ingrowth medium and pre-incubated for 15 hours with a serial 5-folddilution of interferon αn3, interferon β1a and Multiferon™ in 24-wellplates. The following day, the medium was aspirated and 100 microlitresof virus was added to each well at a titre of 100 pfu/well.

After incubation for one hour, the virus inoculum was aspirated and acarboxymethylcellulose overlay containing maintenance medium and theappropriate interferon concentration was added. After four daysincubation, the plates were fixed and stained as described above.

Viral plaques were visible 3 days after pre-incubation of infected cellsfor 15 hours with five-fold dilutions of the interferon. Plaques werethen counted visually and the concentration of the interferon whichinhibited 50% of plaques in each well (IC₅₀) determined. Results wereplotted in Microsoft Excel, and a polynomial of order three was used toapproximate the data and extrapolate IC₅₀ and IC₉₅ values. (Results notshown)

The assay was also carried out in duplicate as described above forMultiferon™ at a viral titre of 54 pfu/well.

Interferons are known to be relatively species specific as the targetfor the interferon is the infected cell rather than the virus itself.The anti-viral activity of Multiferon™ was also assessed in a human cellline, the pulmonary epithelial cell line A549.

Results

Cytopathic Endpoint Assay

The cytopathic effect of SARS-HCoV was evident within 24 hours followinginfection. Infected cells were rounded and exhibited monolayerdestruction.

Complete inhibition using a high viral challenge (10⁴ pfu/well) and highmultiplicity of infection (0.5) was observed for Ribavirin™, and for theMultiferon™ product. At a viral load of 10² pfu/well the CIA₁₀₀ valuewas 5 IU/ml for Multiferon™, with no cytotoxicity observed.

Although Ribavirin™ showed inhibitory activity at all viral titres thiswas only at high concentrations of the drug. Such concentrations showedcytotoxicity and thus Ribavirin™ is not likely to be a clinicallyeffective treatment for severe acute respiratory syndrome (SARS)coronavirus.

In contrast, Multiferon™ did not show any cytotoxicity at thisinhibitory concentration.

Interferon αn3, interferon αn1 and interferon β1b also showed inhibitionof cytopathic effect using this assay. Interferon α2a, interferon α2band interferon β1a did not show significant inhibition (results notshown).

Results are shown for Multiferon™ and Ribavirin™ in Tables 1 and 2below. TABLE 1 Results of the Cytopathic Endpoint Assay for Multiferon ™and Ribavirin ™. (Results not shown for other treatments tested)Concentration at which complete Anti-viral cytopathic Treatment effectCIA₁₀₀ Multiferon ™ 5,000 IU/ml Yes Ribavirin ™ 5,000 μg/ml Yes

TABLE 2 Data obtained for Multiferon ™ and the anti- viral product,Ribavirin ™. (Results not shown for the other treatments tested). VirusLoad Multiferon ™ Ribavirin ™ (pfu/well) (IU/ml) (μg/ml) 1,000 50 5,000100 5 500Plaque Reduction Assay

The Multiferon™ preparation displayed a dose-dependent inhibition ofSARS-HCoV plaque formation. IC₅₀ and IC₉₅ values for Multiferontreatment were 2 IU/ml and 44 IU/ml, respectively. Results are shownbelow for Multiferon™ in Table 3 and in FIG. 1 for a viral titre of 54pfu/well. An EC₅₀ value of 3.16 IU/ml was obtained. TABLE 3 Resultsobtained in the plaque reduction assay for Multiferon ™ at 54 pfu/well.Multiferon ™ Log Multiferon ™ % plaque % plaque Average ConcentrationConcentration reduction reduction plaque (IU/ml) (Log IU/ml) (Well 1)(Well 2) reduction 5000 3.69897 100 100 100 1000 3 100 100 100 2002.30103 100 100 100 40 1.60206 100 100 100 8 0.90309 68.5 75.9 72.2 1.60.20412 40.7 48.1 44.4 0.32 −0.49485 18.5 25.9 22.2 0.064 −1.19382 0 0 0

Interferon αn3 and interferon β1a also showed dose-dependent inhibitionof SARS-HCoV plaque formation in this assay (results not shown).

Example 2

SARS-HCoV, strain Frankfurt-1, kindly provided by the Bernard NotchInstitute, Frankfurt, Germany, was propagated on Vero E6 cells, anAfrican Green Monkey cell line obtained from American Type CultureCollection, Manassas, Va., USA. For titration of the virus, serialdilution of SARS-HCoV were added to Vero E6 cells grown in micro-plateswith Eagle's medium containing 2% foetal calf serum. After 3 days ofculture, cytopathogenic effects were determined microscopically andcytotoxity was then assayed using a calorimetric assay based on themeasurement of lactate dehydrogenase (LDH) activity released from thecytosol of damaged cells (Cytotoxicity detection kit, Roche DiagnosticsGmbH, Penzberg, Germany).

For the antiviral experiments the following four different commerciallyavailable interferon preparations were used: 1) Intron A™, ScheringPlough, USA; 2) Roferon™, Roche, Switzerland; 3) Betaferon™, ScheringAG, Germany and 4) Multiferon™ (Viragen, Fla., USA).

Serial 5-fold dilutions (0.2-31.125 IU/ml) of the interferonpreparations were added to Vero E6 cells in micro-plates which were thenincubated overnight at 37° C. SARS-HCoV was then added at differentconcentrations. (1000, 100 or 10 TCID₅₀) to different sets of interferondilutions, and after a further incubation of 3 days the plates were readmicroscopically, and then by the ELISA LDH cytotoxicity assay.

In a separate set of experiments, the method used by Cinatl et al.(2003) including addition of interferon on two occasions, one day beforeand one day after addition of the virus to the plates, was employed.

In all experiments, controls with 1) virus but not interferon, 2) alldifferent dilutions of the interferons but no virus, and 3) no virus andno interferon were included.

Results

The cytotoxicity (LDH) assay used for determination of SARS-HCoVcytopathogenic effect (CPE) was found to be highly reliable, giving ODvalues in CPE-positive cultures of 1.5-1.8 and in CPE-negative culturesvalues not exceeding 0.2.

Although two of the interferons, Roferon A™ and Multiferon™ showed atendency to increase baseline levels in the cytotoxicity assay, theresult showed no dose-dependent increase in these levels and the ODvalues did not exceed 0.6 in any case. There was no similar tendency forIntron A™ or Betaferon™ The concentration of interferons capable ofdecreasing OD values of virus-infected cultures by 50% (IC₅₀) are shownin Table 4 which shows the results of experiments where IFN was addedeither once (type 1) or twice (type 2) to the cells. TABLE 4 Effect ofvarious interferons on SARS-HCoV replication IC₅₀ Exp. Type 1 IL₅₀ Exp.Type 2 Interferon 10 TCID₅₀ 100 TCID₅₀ 10 TCID₅₀ 100 TCID₅₀ Betaferon110  625 110  190 Multiferon 540 2400 490 2200 IntronA >3.125 >3.125 >3.125 >3.125 Roferon >3.125 >3.125 >3.125 >3.125

IC₅₀ values given as IU of interferon per ml. Slight inhibition ofcytotoxicity was obtained with Roferon™ as well as Intron A™ at thehighest concentrations tested, but the reduction of OD values did notreach the 50% level in any experiment with these interferons.

The outcome of the two different experiments performed were similar,showing that Betaferon™ had the highest antiviral activity (IC₅₀ 50-500IU/ml) followed by Multiferon™ (IC₅₀ 500-2000 IU/ml). Neither Intron A™nor Roferon™ had any clear antiviral activity at the highestconcentrations used in the experiments (3.125 IU/ml). Extrapolation ofresults obtained with the highest concentrations of the IFN preparationsshowed that IC₅₀ levels could be expected to be reached atconcentrations of 10,000-15,000 IU/ml for the latter two types of IFN-α.

Discussion

The present results corroborate earlier findings that IFN-β has anantiviral activity against the SARS-HCoV, that is superior to that ofrecombinant, IFN-β2, interferons (Cinatl et al., 2003). Furthermore, theresults indicate that multi-subtype, natural IFN-α, albeit being lessactive that β-interferon, also has a significant effect on SARS-HCoVreplication. The latter finding agrees with the recent results by Tan etal. (2004) who found, using a plaque reduction assay, that two types ofnatural IFN-α preparations showed strong anti SARS-HCoV activity with apotency that was only slightly lower than that obtained withβ-interferon.

The accumulated evidence now suggests that interferons may have a rolein the treatment of severe acute respiratory syndrome (SARS)coronavirus. The promising results of Loutfy et al. (2003) were obtainedusing a recombinant so-called consensus IFN-α (Infergen) that isbelieved to have effects that are shared by various subtypes of IFN-α.The suggestive clinically beneficial effect of the consensus IFN-α maybe concordant with the presently obtained in vitro results with nIFN-α,but as far as we are aware, no studies on the relative in vitroactivities of nIFN-α and consensus IFN-α have been performed.

Example 3 Anti-Viral Effect of Multi-Subtype Interferon as Compared toIntron A Against Semliki Forest Virus in Vero E6 Cells

Vero E6 cells were seeded in 96-well plate, at a density of 10000 cellsper well. After incubation overnight at 37° C., cells were incubatedwith 100 ul of a serial 10-fold dilution of Multiferon or Intron A(titration range from 1250 IU/ml-2.4 IU/ml). After 24 hours, cells wereinfected with 5000 pfu of Semliki Forest Virus (estimated MOI was 0.1)and further incubated for 48 hours until cytopathic effect was observedin untreated wells. Media was removed from cells, and cells were washedin 1× PBS, then fixed for 10 minutes at room temperature in 4%paraformaldehyde in PBS. Paraformaldehyde was removed and cells werestained with 0.2% crystal violet in 2% ethanol for 10 minutes at roomtemperature. Stained plates were washed and degree of colouration wasquantified at 630 nm using an ELISA reader. Triplicate data is presentedin graph format (FIG. 2).

Results

FIG. 2 demonstrates that Multiferon was found to be effective atprotecting Vero E6 cells from SFV infection over a range ofconcentrations. At 625 IU/ml, the same degree of protection was observedfor both Multiferon and IntronA (results not shown), and an equivalentloss of protection was observed for both products at 39 IU/ml. At allconcentrations in between, Multiferon provided significantly higherprotection that provided by Intron A.

Example 4 Anti-Viral Effect of Multi-Subtype Interferon in Human Cells

Multiferon™ was added prior to addition of the virus. The humanEncephalomyocarditis virus (EMCV) was then used to infect A549 cells andthe effect of Multiferon™ on the cytopathogenicity of EMCV wasdetermined by assessing the interferon concentration required to obtain50% cytopathic effect (CPE) for the human A549 cells. Results are shownin FIG. 3. Cell survival was measured photometrically and results areshown in FIG. 4.

The results show that the Multiferon™ preparation successfully protectedagainst a cytopathic effect on EMCV-infected cells and that the adverseeffect on the host cells did not continue to rise significantly ateffective Multiferon™ concentrations.

FIG. 3 shows the concentrations of Multiferon™ needed to obtain 50%cytopathic effect in the human cells at varying viral titres. As wouldbe expected, a higher viral concentration requires a higher effectiveMultiferon™ concentration.

FIG. 4 shows that Multiferon™ does not have significant adverse celltoxicity effects on human host cells.

Discussion

The results provided show that many interferons are highly effective atinhibiting the activity of the SARS-HCoV. Further, it has been shownthat, in general natural interferons, especially multi sub-typeinterferons, such as Multiferon™, are particularly effective. Moreoverat effective Multiferon™ concentrations, no cytotoxicity is observed.

In tests for anti-viral activity in human cells, Multiferon™ shows agood dose response with cytotoxicity levels which do not rise inproportion to the effective Multiferon™ concentration.

These results indicate that certain interferons such as Multiferon™ arehighly effective therapeutics for the treatment of SARS-HCoV infectionin humans and can be expected to have low levels of adverse effects invivo.

Other groups have studied the efficacy of recombinant interferonproducts against SARS CoV. Stoher et al demonstrated significant butincomplete activity of Intron A at a concentration of 1000-5000 IU/ml oncells infected with a multiplicity of infection (MOI) of 0.001 plaqueforming units per cell in a cytopathic endpoint assay. However, theresults presented show that Multiferon™ used at the low dose of 5 IU/mlcompletely protected cells from SARS-HCoV infection at a MOI of 0.005plaque forming units per cell, five times greater than the MOI used inthe Intron A™ experiments. Furthermore, 50 IU/ml of Multiferon™protected cells from SARS-HCoV infection at a MOI of 0.05, 50 timesgreater than the MOI utilised in the Intron A™ studies. Finally, in ourstudies, concentrations of Intron A™ or Roferon™ up to 100000 and 500000IU/ml, respectively, failed to fully protect cells from SARS-HCoVinfection.

Whilst Stoher et al. claim that doses of up to 3.6×10⁷ IU/ml have beeninfused intravenously, and that serum concentrations of at least 500IU/ml are achievable after intramuscular injection, the serum titrewould only reach this level for a short period of time, and intravenousinfusion has highly toxic implications. Taken together with the resultsdescribed, this supports the significant superiority of naturalmulti-subtype interferon products, in particular Multiferon™, overrecombinant IFN alpha2 preparations.

All publications and patent documents referred to herein areincorporated by reference in their entirety. Although the invention hasbeen described in connection with specific examples, it should beunderstood that the invention should not be unduly limited to suchexamples. Specifically, it will be understood by one skilled in the artthat various modifications to and variations of the invention asdescribed herein may be made without departing from the scope of theinvention.

REFERENCES

-   Al-Jabri et al. In Mahy, B W J and Kangro, H O eds. Virology Methods    Manual, Academic Press Ltd, London (1996). 293-356-   Cinatl, J et al. Lancet 362 (9380) 293-294-   Crotty, S. et al. Nat. Med 6 1375-1379-   Goodbourn, S. E. Y., et al. (2000). J. Gen. Virol. 81 2341-2364.-   Lee, N. et al. N. Engl. J. Med. 348(20) 1986-1994-   Loutfy, M. R. et al., JAMA 290(24) 3251-3253-   Poutanen, S. M. et al. (2003) New Engl. J. Med. 348 (20) 1995-2005-   Sidwell, R. W. et al. Antimicrob. Agents Chemother. 31 1130-1134-   Stoher, U. et al. (2004). Journal of Infectious Diseases. 189:1164-7-   Tam, R. C. et al. Antivir. Chem. Chemother. 12 (5) 261-272-   Tan, E. L. C. et al. (2004). Emerging infectious diseases. 10(4)    581-586-   Weck, P. K. et al. 1981. J. Gen. Virol. 57 233-237-   Weiss, R. C. & Oostrom-Ram, T. Vet Microbiol. 20 255-265

1. A method of treating or preventing severe acute respiratory syndrome(SARS) coronavirus (SARS-HCoV) infection, the method including the stepof administering a therapeutically useful amount of an interferon to asubject in need of treatment, wherein the interferon is a multi-typeinterferon consisting of interferon alpha (IFNα), interferon αn1,interferon αn3 or interferon β1b.
 2. A method as claimed in claim 1wherein the interferon is derived from human cells.
 3. A method asclaimed in claim 1 wherein the interferon is recombinant.
 4. A method asclaimed in any claim 1 wherein the interferon is an isolated interferon.5. A method as claimed in claim 1 wherein the interferon ismulti-subtype, human alpha-interferon derived from white blood cellscommercially available as Multiferon™.
 6. A method as claimed in claim 1wherein the subject is human.
 7. (canceled)
 8. (canceled)
 9. (canceled)10. (canceled)
 11. (canceled)
 12. A method of treating infection withsevere acute respiratory system (SARS) coronavirus (SARS-HCoV), themethod including the step of administering a therapeutically usefulamount of an interferon to a subject in need of treatment along with atherapeutically useful amount of a suitable anti-viral compound, whereinthe interferon is a multi-subtype interferon consisting of interferonalpha (IFNα), interferon αn1, interferon αn3 or interferon β1b.
 13. Amethod as claimed in claim 12 wherein the interferon is themulti-subtype, human alpha-interferon derived from white blood cellscommercially available as Multiferon™.
 14. A method as claimed in claim12 wherein the anti-viral compound is ribavirin.
 15. A method as claimedin claim 12 wherein the subject is human.
 16. A combined medicamentcomprising an interferon and an anti-viral compound for use in thetreatment or prevention of infection with a severe acute respiratorysystem (SARS) coronavirus (SARS-HCoV), wherein the interferon is amulti-subtype interferon consisting of interferon alpha (IFNα),interferon αn1, interferon αn3 or interferon β1b.
 17. A combinedmedicament as claimed in claim 16 wherein the interferon is themulti-subtype, human alpha-interferon derived from white blood cellscommercially available as Multiferon™.
 18. A combined medicament asclaimed in claim 16 wherein the infection is infection of a human. 19.An assay method for determining the efficacy of a candidate agent in thetreatment of infection with a severe acute respiratory system (SARS)coronavirus (SARS-HCoV), the assay method including the steps of:incubating cells infected with coronavirus in the presence of thecandidate agent, determining the degree of inhibition of the cytopathiceffect of the virus on the cells, and comparing the degree of inhibitionobtained using the candidate agent with the degree of inhibitionobtainable with incubation with a multi-subtype interferon consisting ofinterferon alpha (IFNα), interferon αn1, interferon αn3 or interferonβ1b, or a product based on these inteferons.
 20. An assay as claimed inclaim 19 wherein the interferon is the commercially availablemulti-subtype interferon alpha composition Multiferon™.
 21. An assay asclaimed in claim 19 wherein the infection is infection of a human. 22.(canceled)
 23. (canceled)
 24. (canceled)
 25. A multi-subtype interferonconsisting of interferon alpha (IFNα), interferon αn1, interferon αn3 orinterferon β1b for use in the treatment or prophylaxis of a severe acuterespiratory syndrome (SARS) coronavirus (SARS-HCoV) infection.
 26. Amulti-subtype interferon as claimed in claim 7 wherein the interferon ishuman alpha-interferon derived from white blood cells commerciallyavailable as Multiferon™.
 27. A multi-subtype interferon as claimed inclaim 7 wherein the interferon is recombinant interferon.